Culture of goblet cells

ABSTRACT

The invention encompasses isolation, culture and characterization of goblet cells in vitro from mammalian conjuctiva. Goblet cells can be cultured from conjunctiva of such mammals as, e.g., humans, rats, mice, rabbits and the like. In another aspect of the invention, the culture of goblet cells has a concentration of pure goblet cells of 10% or greater. In a further embodiment, the invention comprises an immortalized goblet cell line.

CROSS REFERENCE TO RELATED APPLICATIONS

This present application is a divisional application of U.S. applicationSer. No. 10/398,574 which was filed on Apr. 7, 2003 entitled CULTURE OFGOBLET CELLS which a 35 U.S.C. §371 filing of International ApplicationNo. PCT/US01/31485 was internationally filed on Oct. 5, 2001 and whichfurther claims the priority of U.S. Provisional Application No.60/238,220, filed on Oct. 5, 2000, the whole of which are each herebyincorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The work leading to the invention received support from the UnitedStates federal government under grant no. NIH EY 09057. Therefore, thefederal government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

The epithelium comprising the conjunctiva is classified as anon-keratinizing stratified squamous epithelium consisting of severallayers (Gipson, 1994). Goblet cells, highly specialized epithelial cellsare located in the apical surface of the conjunctiva, interspersed amongthe layers of stratified epithelium (Wei et al.; Geggel et al.). Thesecells are readily identified by their extensive apical accumulation ofsecretory vesicles (Jeffery et al.; Huang et al.) and can occur eithersingly as in humans and other mammals (Kessing; Latkovic; Tseng et al.)or in clusters as found in the conjunctiva of adult rats (Srinivasan etal.). Irrespective of species, goblet cells are primarily responsiblefor the secretion of the inner mucous layer of the tear film, whichprovides a physical and chemical barrier to protect the ocular surfacefrom dryness or other deleterious environments and/or a variety ofnoxious agent. (Lamberts; Nichols et al.; Gibbons; Lemp et al.) In thisregard, goblet cells synthesize, store and secrete high molecular weightglycoproteins referred to as mucins, which upon secretion have theability to hydrate and gel, producing a protective scaffolding over theocular surface. (Chao et al.; Steuhl) Maintenance of this covering isessential to the health of the corneal and conjunctival surface.Inability or interference in the ability of goblet cells to secretenormal levels of mucin can lead to pathological abnormalities within theconjunctiva. Mucin deficiency often results as a consequence of ocularcicatricial pemphigoid, Steven Johnson syndrome, alkali burns andneurotrophic keratitis whereas overproduction of mucin due to excessivegoblet cell secretion or proliferation is thought to be mediated byactivated T-cells and macrophages and by a chronic conjunctivitis suchas atopic keratoconjunctivitis. (Lemp, 1973; Tseng, Ophthalmol., 1984;Gilbard et al.; Lemp, 1992) These diseases and their sequellae caneventually lead to deterioration of the ocular surface.

Because the importance of the goblet cell in maintaining the integrityof the ocular surface is well recognized, a large number of structural,ultrastuctural and histochemical studies have been performed on theconjunctival epithelium in a variety of species (Latkovic; Steuhl;Setzer et al.; Moore et al.; Oduntan; Breithnach et al.). Data derivedfrom these studies have provided valuable information regarding thedevelopment, subsequent appearance, location and function of gobletcells within the conjunctiva. In addition, they have providedinformation concerning the influence of environmental factors, chemical,toxin and disease upon these same goblet cell parameters.

Previous reports of systems developed to culture goblet cells in vitroare limited. Goblet cell cultures derived from airway epithelia ofhamsters, rats and humans (Wu et al., 1985; Kaartinen et al.; Wu et al.,1990) have been in use for several years. By comparison, the developmentof systems to culture conjunctival goblet cells is still in its infancy.Among the methods which have been used to study these cells include:sectioning of conjunctival tissue combined with a battery ofhistochemical staining; immunocytochemical localization; transmissionelectron microscopy and in situ hybridization (Huang et al.; Greiner etal.; Allansmith et al.; Kinoshita et al.); histochemical staining ofwhole-mounted tissue (Huang et al.; Tseng et al., Ophthalmol. Vis. Sci.,1984); PAS staining of filter paper strips applied to the conjunctivalsurface (Adams); phalloidin labeling of excised conjunctiva (Gipson,1997) and neutral protease removal of viable sheets of conjunctivalepithelium (Geggel et al.) and growth of conjunctival cells on varioussubstrata including natural extracellular matrix components, fibroblastfeeder layers and on collagen and matrigel (Sun et al.; Rheinwald etal.; Tsai et al.). These systems are limited in that they yield indirectinformation. The limitations result from the information often beingextrapolated from studies using whole-mounted or sectioned conjunctivaltissue, or from being derived from intestinal and tracheal neoplasticcell lines, which mimic only select functions of goblet cells.Conjunctival cells have been grown from a variety of tissues includinghuman, but no reproducible, characterized system by which goblet cellscan be propagated has been reported.

Successful and consistent isolation and culture of goblet cells withoutaltering their phenotype and/or function has been limited. Inparticular, normal human diploid cells have a limited proliferativelifespan in culture. In the past, it was necessary to use a variety ofcomplex culture media as well as artificial matrices in order for thecells to attach. These techniques, however, would not always insuregrowth, propagation and preservation of cellular function. Therefore,there remains a need for a reliable method for culturing mammaliangoblet cells.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to the isolation and subculturingconjunctival mammalian goblet cells, which exhibit morphological,histochemical, immunocytochemical, and biochemical markers indicative ofgoblet cells in vivo. The culture of goblet cells in accordance with thepresent invention provides a more effective in vitro test format, interalia, while retaining the original phenotypic characteristics associatedwith goblet cells in vivo.

In an embodiment of the invention, a culture of goblet cells isolatedfrom mammalian conjunctival tissue has a concentration of pure gobletcells of 10% or greater. Conjunctival tissue of the present inventionincludes the fornical region and the nictitating membrane. Inparticular, human goblet cells are obtained from the fornical region.The mammal of the present invention can include, inter alia, humans,rats, mice, rabbits, cats, dogs, sheeps, goats, cows, and pigs.

In a further embodiment of the invention, the concentration of gobletcells maintained in culture is preferably 10%-30%; more preferably30%-50%, yet more preferably 50%-70%, still more preferably 70%-90%,most preferably 90%-95%, and still most preferably 95%-100%.

In another embodiment, the invention comprises a method of producing aculture of goblet cells, which has a concentration of pure goblet cellsof 10% or greater. In one aspect, the method comprises providing anexplant of conjunctival mammalian tissue; culturing the explant in agrowth medium; allowing the explant to grow until cell growth in theform of nodules is observed around the explant; removing the explant,leaving said nodules in the growth medium; and then allowing the cellsfrom the nodules to grow to form the culture of goblet cells. In anotheraspect of the method, the cells growing separately from the nodules areremoved.

In another aspect of the method, the concentration of goblet cellscomprised in a culture is preferably 10%-30%; more preferably 30%-50%,yet more preferably 50%-70%, still more preferably 70%-90%, mostpreferably 90%-95%, and still most preferably 95%-100%.

In a further aspect of the method, the conjunctival mammalian tissuecomprises the fornical region and the nictitating membrane. Inparticular, human conjunctival tissue comprises tissue from the fornicalregion. In a further aspect of the method, the mammal is selected fromthe group consisting of human, rat, mouse, rabbit, cat, dog, sheep,goat, cow, and pig.

In a further embodiment, the invention comprises a culture of gobletcells with an extended lifespan made by the method of the presentinvention. In one aspect, the goblet cells of the invention areimmortalized goblet cells, which are made from the method of making aculture of goblet cells of the present invention. This culture of gobletcells has a concentration of 10% or greater of pure goblet cells. In aparticular aspect, the invention comprises an immortalized goblet cellline, which has a concentration of 100% goblet cells.

In yet another embodiment, the immortalized goblet cells as well asgoblet cells made from the method of culturing described herein may beproduced into a kit with instructions for use for examiningmucin-associated effects. The kit may be used, for example, but notlimited to screening various toxic compounds and consumer products,diagnosing for conditions associated with mucin deficiency, studying forallergic reactivity of various foreign substances and quantitating theamount of mucin.

In a further embodiment, the invention comprises a method of treating apatient suffering from conditions associated with conjunctival mucindeficiency. In one aspect, the method includes identifying a patientsuffering from conditions associated with conjunctival mucin deficiency;providing a therapeutic composition in a pharmaceutically acceptableform for administration comprising goblet cells of the presentinvention; and administering to the patient a therapeutically effectiveamount of the composition. In a further aspect of the method oftreatment, the goblet cells may be obtained from the culture of gobletcells of the invention. In another aspect, the goblet cells used totreat a patient may originate from the same patient to avoid rejectionand/or deleterious autoimmune responses. In a further aspect of themethod, the pharmaceutically acceptable form for administration mayinclude, e.g., autograft transplantation, eye drops, corneal bandage,ointments, and topical treatment. In another aspect, the method includesconditions associated with conjunctival goblet cell mucin deficiency,for example, but not limited to, lacerated corneas, ocular cicatricialpemphigoid, Steven Johnson syndrome, alkali burns, and neurotrophickeratitis.

BRIEF DESCRIPTION OF THE DRAWINGS

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof and from theclaims, taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a phase contrast photomicrograph showing a representativeexplant culture of rat conjunctival tissue grown in RPMI-1640 culturemedium supplemented with 10% FBS. Cells are seen growing out of thetissue plug within days of establishment of the culture. After 7-20days, the tissue plug and its cells are surrounded by a ring of nodules(Magnification 40×);

FIGS. 2A, 2B, 2C, and 2D show photomicrographs illustrating the growthpatterns of conjunctival explants in vitro. When examined closely, eachnodule (arrow) is the apparent source of numerous goblet cells, whichtraverse the underlying epithelial cells (FIG. 2A). The single cellsmove along the epithelium until they find an empty space and adhere tothe bottom of the culture flask exhibiting a cobblestone morphology(FIG. 2B). Goblet cells contain tiny translucent droplets, which aresimilar in appearance to mucin on their surface (FIG. 2C). As gobletcells grow in vitro, so do the droplets. They often appear as if themucin droplets are released off and secreted into the cell medium (FIG.2D) (Magnification 100×);

FIG. 3 is an immunocytochemical reaction of the nuclear antigen Ki-67with cultured goblet cells. Goblet cells in primary and passaged culturereact positively with Ki-67 which selectively stains the nuclei of cellsactively engaged in proliferation as shown here (Magnification 250×);

FIGS. 4A, 4B, and 4C depict histochemical reactivity of cultured gobletcells to AB-PAS. Goblet cells stain intensely with AB/PAS indicating thepresence of both acidic (blue) and neutral (pink) glycoconjugatesassociated with the cells (FIG. 4A) (Magnification 100×). Goblet cellswhich appeared to contain secretory droplets on their surface alsoreacted strongly to AB/PAS staining a pinkish color while the dropletsthemselves stained bright red indicating the presence of with a neutralmucin product (FIG. 4B) (Magnification 600×). As a positive control,secretory products present in goblet cells of the conjunctiva reactedpositively to AB/PAS (epi, epithelium) (Magnification 200×);

FIGS. 5A, 5B, 5C, and 5D depict Lectin histochemistry confirming thatrat conjunctival goblet cells were associated with UEA-1, whichrecognizes the 1-fucose moiety of glycoproteins in the secretorygranules of goblet cells (FIG. 5A) and the secretory product in gobletcells located in the conjuntiva (FIG. 5B). In addition, both primaryculture goblet cells (FIG. 5C) and goblet cells present in sections ofrat conjunctiva (FIG. 5D) were reactive to HPA, which recognizes1-galactosamine within their secretory granules (epi, epithelium)(Magnification 200×);

FIGS. 6A and 6B depict an immunocytochemical localization of MUC5ACindicating that secretory products of cultured goblet cells (FIG. 6A)and goblet cells located in conjunctival tissue (positive control) (FIG.6B) contain mucin. Almost all cells were positive for MUC5AC whenvisualized using fluorescence microscopy (Magnification 100×);

FIGS. 7A and 7B depict cultured goblet cells (FIG. 7A) and goblet cellsin conjunctival sections (FIG. 7B) displaying intense immunocytochemicalstaining for cytokeratin-7 (CK-7), a specific marker of intermediatefilaments associated exclusively with goblet cells (Magnification 200×);

FIGS. 8A and 8B depict an immunocytochemical localization ofcytokeratin-4 (a negative control). Primary cultures of goblet cellsisolated by a modified explant culture technique were mostly negativefor cytokeratin-4. An occasional stratified epithelial cell isinfrequently present on top of the underlying goblet cells (FIG. 8A).The underlying goblet cells are shown in (FIG. 8B) (Magnification 100×);

FIGS. 9A and 9B depict an immunocytochemical reactivity of goblet cellsto the muscarinic receptor subtype 3 (M₃) shown in a primary, mixedconjunctival explant culture (FIG. 9A) and in rat conjunctival tissue(FIG. 9B). Only goblet cells stain for the M₃ receptor (Magnification100×);

FIGS. 10A and 10B depict the transmission electron micrographs of tworepresentative cultured goblet cells sectioned en-face. These cellsdisplay degrees of cell polarity and the presence of numerous, intactsecretory granules (Magnification 6000×);

FIGS. 11A and 11B depict the Western blot analysis of UEA-1 containinghigh molecular weight glycoprotein (FIG. 11A) and MUC5AC in culturedgoblet cells and homogenized conjunctival epithelium (FIG. 11B);

FIG. 12 depicts a phase contrast micrograph showing a representativeprimary culture of human goblet cells (Magnification 200×);

FIGS. 13A and 13B depict histochemical reactivity of cultured humangoblet cells positive for AB-PAS. Cultured cells stained with AB/PASdisplayed both acidic (blue) and neutral (pink) glycoconjugatesassociated with the cells. In addition, as a positive control, gobletcells within conjunctival tissue reacted positively to AB/PAS(Magnification 200×);

FIG. 14 depict an immunocytochemical staining for cytokeratin 4 in aculture of human goblet cells. Cytokeratin 4 is a specific marker ofstratified squamous epithelial cells, which the staining indicates thatrelatively few of these contaminating cell types are present in humangoblet cell cultures (Magnification 200×);

FIG. 15 depicts an Immunocytochemical staining of cultured human gobletcells that are positive for cytokeratin 7, which is a specific marker ofintermediate filaments associated specifically with goblet cells(Magnification 200×);

FIGS. 16A and 16B depict a positive histochemical reactivity to thelectin Helix pomatia agglutinin (HPA) in cultured human goblet cells andin goblet cells within human conjuctival tissue. HPA recognizes1-galactosamine within the secretory granules of goblet cells(Magnification 200×);

FIGS. 17A and 17B depict an immunocytochemical staining of culturedhuman goblet cells that are positive for HPA and cytokeratin 7. Thisshows dual localization of HPA and cytokeratin 7 in both cultured gobletcells and goblet cells located in human conjunctival tissue. Cytokeratin7 is identified by fluorescein labelling (green) and HPA by rhodaminelabelling (red) (Magnification 200×);

FIGS. 18A and 18B depict an immunocytochemical localization of thegoblet specific mucin MUC5AC. The cultured goblet cells are positive forMUC5AC, including goblet cells of human conjunctiva (Magnification200×); and

FIG. 19 depicts a positive immunocytochemical staining for MUC5AC incultured goblet cells. MUC5AC is contained within the secretory granulesof human goblet cells as shown (Magnification 500×).

DETAILED DESCRIPTION OF THE INVENTION

A “culture of goblet cells” designates a culture of goblet cells havingat least a concentration of 10% or more of goblet cells. In a furtherembodiment, the concentration of goblet cells maintained in culture ispreferably 10%-30%; more preferably 30%-50%, yet more preferably50%-70%, still more preferably 70%-90%, most preferably 90%-95%, andstill most preferably 95%-100%.

A “primary culture” or “non-immortalized culture” designates a cultureof goblet cells, which can be cultured for a limited time without losingtheir original differentiation characteristics.

An “immortalized cell” designates cells which have been geneticallyengineered, allowing them to multiply indefinitely.

A culture with an “extended lifespan” has the capability of successiveculturing, preferably at more than three passages, while retainingoriginal differentiation markers of cells.

A “passage” designates the process consisting of taking an aliquot of aconfluent or saturation culture of cells, inoculating a fresh mediumwith an aliquot of cells, and culturing the cells until confluence orsaturation is obtained. The cells are thus traditionally cultured bysuccessive passages in fresh media. Unlike the present invention, aftersuccessive passages of culture, cells ordinarily tend to lose theiroriginal differentiation characteristics. Human diploid cells, inparticular, have a limited proliferative lifespan in culture. Thepresent invention comprises a method of successive passaging of culturedgoblet cells, which retains the cells original phenotypiccharacteristics in vivo. The culture of the present invention may bepassaged at least three times or more.

An “original phenotypic characteristics” of goblet cells designate thepresence of particular markers representative of goblet cells in vivo:e.g., positive staining for alcian blue/Periodic acid Schiff's (AB3/PAS)reagent, cytokeratin 7, the lectins Ulex europaeus agglutinin-I (UEA-I)and helix pomatia agglutinin (HPA), MUC5AC and M₃ muscarinic receptor;negative staining for cytokeratin 4, M₁ muscarinic receptor and banderiasimplicifolia lectin.

The invention relates to a method by which one can simply andreproducibly isolate and subculture conjunctival goblet cells, whichexhibit morphological, histochemical, immunocytochemical, andbiochemical markers indicative of goblet cells in vivo and retain thesemarkers upon subcultivation. The culture of goblet cells in accordancewith the present invention provides a more effective in vitro testformat while retaining the original phenotypic characteristicsassociated with goblet cells in vivo. Suitable sources of goblet cellsfor culture by the present method include conjunctival tissues frommammals such as, but not limited to, humans, rats, mice, rabbits, cats,dogs, sheep, goats, cows, and pigs. In a preferred embodiment of thepresent invention, the conjunctival tissue may comprise the fornicalregion and the nictitating membrane.

Evidence is presented herein that primary cultures of goblet cells canbe isolated from fragments of mammalian conjunctiva using a modifiedexplant culture system. Primary and passaged cultures of mammaliangoblet cells obtained from the fornical region of the conjunctiva andfrom the nictating membrane, reacted positively with Alcianblue/Periodic acid Schiff's reagent (AB/PAS) and with the goblet cellspecific lectins, Ulex europaeus agglutinin-1 (UEA-1) and Helix pomatiaagglutinin (HPA), similarly to their counterparts in vivo. Theypresented positive, selective staining for the intermediate filament,Cytokeratin-7 and for the mucin, MUC5AC, selective markers for gobletcells in vivo, and secreted mucin into their culture medium. Moreover,these same markers persisted after subcultivation.

Isolation, in vitro culture, and characterization of goblet cells wereperformed using the following materials and methods. The method of theinvention is described with reference to the following, non-limitingexemplary protocol using both human and rat goblet cells. At the sametime, those of ordinary skill in the art of cell culture will appreciatethat certain minor substitutions can be made (e.g., changes to theculture medium), so as to adapt the present goblet cell culture systemfor use with various mammalian tissues.

Exemplary Materials and Methods Materials

RPMI-1640 culture medium, L-glutamine, penicillin/streptomycin, Hank'sBalanced Salt Solution, trypsin-EDTA solution were obtained fromBioWhittaker (Walkerville, Ill.), fetal bovine serum from HycloneLaboratories (Logan, Utah). Falcon tissue culture flasks, pipettes, andother routine plastics were obtained from Becton Dickson Labware(Franklin Lakes, N.J.). Glass coverslips were from VWR Scientific (SanFrancisco, Calif.). Lab Tek chamber slides were obtained from NUNC, Inc.(Naperville, Ill.). Monoclonal antibody against Cytokeratin 7 (CK7) wasfrom ICN (San Francisco, Calif.) and against Ki-67 was from NovocastraLabotatories, Ltd (New Castle Upon Yyne, UK). Ulex europeus agglutininlectin, (UEA-1) and Helix pomatia agglutinin lectin (HPA) directlyconjugated with fluorescein isothiocyanate (FITC) or Texas Red wereobtained from Pierce, (Rockford, Ill.). Banderia simplicifolia lectin(BS-1) conjugated to FITC was obtained from Vector Laboratories(Burlingame, Calif.). Polyclonal antibodies against M₁ and M₃acetylcholine receptor (AchR) subtypes were obtained from Research andDiagnostics Laboratory (Berkeley, Calif.). All other chemicals unlessotherwise specified were obtained from Sigma (St. Louis, Mo.). Thecytokeratin 4 antibody was a gift of Dr. James Zieske, Schepens EyeResearch Institute, (Boston, Mass.). Dr. Ilene Gipson, Schepens EyeResearch Institute, (Boston, Mass.) provided antibodies to rat and humanMUC5AC. Dr. Marsha Jumblatt, University of Louisville School ofMedicine, (Louisville, Ky.) provided the antibody to human MUC5AC.

The following examples are presented to illustrate the advantages of thepresent invention and to assist one of ordinary skill in making andusing the same. These examples are not intended in any way otherwise tolimit the scope of the disclosure.

Methods

Isolation and culture of cells. All removal of tissue and subsequentmanipulations of animals used in this study conformed to the guidelinesestablished by the ARVO Statement for the Use of Animals in Ophthalmicand Vision Research and were approved by the Schepens Eye ResearchInstitute Animal Care and Use Committee. Male Sprague-Dawley ratsweighing between 250 and 300 g were used in this study and were obtainedfrom Taconic Farms (Germantown, N.Y.). Rats were anesthetized for 1minute in CO₂, decapitated and both eyes surgically removed.Conjunctival tissue, more specifically the nictating membranes and/orfornix, were excised and immediately placed into Hank's Balanced SaltSolution containing 3× penicillin/streptomycin (300 μg/ml). Tissue wasfinely minced into 1-2 mm³ pieces that were anchored onto either scoredculture dishes or onto glass coverslips placed within 6-well culturedishes. The culture dishes contained just enough medium to cover thebottom of the dish (e.g., 400-500 μl), so that the tissue would receivenutrients via surface tension. (Otherwise, if the tissue was submerged,it became necrotic.) Cell medium used to feed explants and culturegoblet cells consisted exclusively of RPMI −1640 medium supplementedwith 10 heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine and100 μg/ml penicillin-streptomycin. Explants were refed every two dayswith the medium described above and were grown under routine cultureconditions of 95% O₂:5% CO₂ at 37° C.

Cells were permitted to grow from the explant plug until evenly spacednodules were evident forming a circular pattern around the explant plug(see, e.g., FIG. 1). The explant plug was then removed and discarded,while leaving the nodules to grow goblet cells. At this juncture, allcells that grew outside this circular perimeter, or cells that grewseparately from the nodules, were removed by scraping the bottom of thedish with a rubber policeman, and discarded. Goblet cells and cells ofneural lineage then were observed to grow from these nodules, eventuallycovering the remainder of the culture vessel. Some cultures weretrypsinized and passaged after reaching confluence. Cells were routinelypassaged by trypsinization of confluent, adherent cells with 0.05%trypsin, 0.53 mM EDTA, pH 7.4.

Histochemistry. Cells were fixed with 100% methanol and processed forAB/PAS (Sheehan et al.) and lectin histochemistry. Goblet cells examinedfor lectin histochemistry were grown on either chamber slides, glasscoverslips or plastic tissue culture wells, rinsed in PBS, and fixed in100% methanol for 15 min at room temperature before they were returnedto fresh PBS. Fixed cells were incubated in blocking buffer whichconsisted of 1% BSA and 0.2% Triton X-100 in PBS for 30 minutes at roomtemperature. Cells then were incubated for 1 h at room temperature witheither UEA-1 conjugated directly to FITC diluted 1:100 in PBS, BS-1conjugated to FITC diluted 1:200 or HPA conjugated to Texas Red anddiluted 1:100 in PBS.

Immunocytochemistry. Methanol-fixed cells were examined for the presenceof Cytokeratins 4 and 7 and MUC5AC. Slides with cultured goblet cellswere incubated for 30 minutes at room temperature in blocking bufferthat contained 1% BSA and 0.2% Triton-X in PBS. Cells were thenincubated with the following dilutions of primary antibodies for 1 hourat room temperature. Antibody to Cytokeratin 7, which recognizes agoblet cell specific keratin, was diluted 1:15 in PBS. Antibody toCytokeratin 4, specific for stratified, squamous, non-goblet epithelialcells, was diluted 1:10 in PBS. Antibody to rat MUC5AC, specific formucin produced by goblet cells, was diluted 1:2000 and 1:4000 in PBS.Antibody to human MUC5AC was diluted 1:1000 in PBS. In order toinvestigate the proliferation profile of cultured goblet cells, antibodyto human Ki-67 nuclear antigen was diluted 1:100 in PBS. For muscarinicreceptor subtypes M₁ and M₃, methanol-fixed cells were incubated inblocking buffer that contained 1.5% normal goat serum and 0.2% TritonX-100 in PBS for 30 minutes at room temperature. Their respectiveantibodies were each diluted 1:2000 in PBS and incubated overnight at 4°C. The secondary antibodies, conjugated to either FITC or rhodamine werediluted 1:200 in PBS and incubated for 1 h at room temperature. Slides,coverslips or dishes were washed 3 times in PBS after which coverslipswere mounted with a media containing 100 mM Tris, pH 8.5, 25% glycerol,10% polyvinyl alcohol, and 2.5% 1,4-diazobicyclo-[2.2.2]-octane. Cellswere viewed using a Nikon Eclipse TE 300 inverted phase contrastmicroscope equipped for fluorescence while cells adherent to glasscoverslips or microscope slides were visualized with a Nikon Eclipse E800 fluorescence microscope. Negative controls consisted of substitutingPBS for the primary antibody. Additional positive controls includedfrozen and/or fixed sections of rat conjunctiva containing prominentgoblet cells.

Transmission Electron Microscopy. Cell-conditioned medium was removedfrom confluent cultures of goblet cells after which monolayers werewashed with cacodylate buffer pH 7.3. Cells were fixed with cacodylatebuffered Karnovsky's solution, post-fixed in 1% osmium tetroxide andembedded in epon according to standard transmission electron microscopytechniques. Thin sections, mounted on copper grids, were stained withlead citrate and examined with a Philips 410 transmission electronmicroscope (Philips, Eindhoven, The Netherlands).

Measurement of goblet cell mucin secretion. Cell-conditioned medium wascollected at various time points following culture (48 hrs, 72 hrs) inorder to measure the amount of mucin released by goblet cells. Theamount of high molecular weight glycoconjugate, an index of mucinsecretion, was determined by ELLA using a biotinylated lectin, UEA-1,known to react with specific carbohydrates present in terminal sugars onmucins synthesized, stored and secreted by goblet cells 21. The ELLA wasperformed following the manufactor's protocol (Pierce, Rockford, Ill.).Biotinylated UEA-1 was used at 2 μg/ml, strepavidin conjugated toalkaline phosphatase was used at 1 μg/ml and the substrate p-nitrophenylphosphate used at 2.5 mM. A 250 μl aliquot of the cell-conditionedmedium was placed on a MaxiSorb titer microplate (Nalge, NUNC, Inc.Naperville, Ill.), and dried overnight at 40° C. Non-specific bindingsites were blocked with 3% BSA, 0.05% Tween-20, and 0.15 M NaCl in 0.25mM Tris-HCl (pH 7.5). Wash buffer contained 0.3% BSA, 0.05% Tween-20 and0.15 M Nacl in 0.25 mM Tris-HCl (pH 7.5). The amount of UEA-1 detectableglycoconjugates in the goblet cell conditioned media were determined induplicate using a microplate reader model MR 700 (Dynatech Labs, WestSussex, UK). A standard curve was constructed using bovine submaxillarygland mucin.

Electrophoresis and immunoblotting. Forty-eight hour oldcell-conditioned medium was removed from young, middle-aged, old andpooled cultures of goblet cells and stored at 4° C. The remaining cellswere scraped and collected into homogenization buffer containing 30 mMTris-HCl, pH 7.5, 10 mM EGTA, 5 mM EDTA, 1 mM DTT, 10 mg/ml PMSF and 5units/ml aprotinin. Cells were further lysed by sonication and whenevernecessary, by freeze-thawing of the cell pellet. To determine thepresence of MUC5AC, a goblet cell-specific mucin, and the glycoconjugaterecognized by UEA-1, proteins present in goblet cell-conditioned mediumand cell lysates were separated by SDS-PAGE using 6% gels andtransferred to nitrocellulose membranes as described by Towbin et al. Tomeasure UEA-1 detectable glycoconjugates, the membranes were blockedovernight at 4° C. in 5% dried milk in TBST consisting of 10 mMTris-HCl, pH 8.0, 500 mM NaCl, and 0.05 % Tween-20 and then incubatedwith biotinylated UEA-1 (1:100) for 1 h at room temperature. Thenitrocellulose membranes were washed three times with TBST and thenincubated with a 1:2500 dilution of horseradish peroxidase-labeledstrepavidin in TBST for 1 h. The membranes were washed three times afterwhich the UEA-1 reactive glycoconjugates were visualized using theenhanced chemiluminescence method. Homogenized adult rat conjunctivaltissue was used as a positive control. In order to detect MUC5AC,membranes were blocked for 1 h as described above and incubated withanti-human MUC5AC antibody (1:500) in 5% dried milk overnight at 4° C.(Jumblatt et al.). The membranes were washed, then incubated for 1 h atroom temperature with a secondary antibody conjugated to horseradishperoxidase in 5% dry milk. They were washed and developed using theenhanced chemiluminescence method as before.

ELISA analysis of human cultured goblet cells using MUC5AC antibody.Materials that were used include the following: Primary antibody MUC5AC(NeoMarker Cat#45 ml) solution adjusted to an appropriate concentration(approximately 2 μg/ml) with wash buffer; anti-mouse IgG-HRP, which wasadjusted to a concentration of approximately 1 μg/ml with wash buffer;bovine submaxillary Mucin type I (Sigma) standard solution at 10-200μg/ml in coating buffer; a coating buffer consisting of 0.1M sodiumbicarbonate buffer at pH 9.2; TBST at 25 mMTris, 150 mM NaCl, pH7.5,0.05% Tween® 20; blocking buffer consisting of TBST and 3% crystallineBSA (Sigma); wash buffer consisting of TBST, 0.3% crystalline BSA(Sigma); substrate solution of dissolved tablet of O-phenylenediamineDihydrochoride (OPD) in 0.2 M dibasic sodium phosphate, and 0.1 M citricacid solution followed by the addition of 4 μl of 30% H₂O₂ in a finalvolume of 10 ml; and titer microplate MaxiSorb, Nalge NUNC.

The method of ELISA analysis included adding 100 μl of mucin standardsolution, 100 μl of cells lysated, or 250 μl aliquot of the culturemedium on a titer microplate (MaxiSorb; Nalge NUNC) and dried overnightat 40° C. Each well were rinsed with 3×200 μl of wash buffer. 200 μl ofblocking buffer were added to the well and incubate for 30 minutes at37° C. Each well were rinsed with 3×200 μl of wash buffer. 100 μl ofprimary antibody MUC 5AC solution were added and incubated for 1 hour at37° C. Each well was rinsed with 3×200 μl of wash buffer. 100 μl ofanti-mouse IgG-HRP were added and incubated for 1 hour at 37° C. Eachwell were rinsed with 3×200 μl of wash buffer. 100 μl of the substratewere added to each well and performed colorimetric reaction.

EXAMPLE I Growth and Morphology of Goblet Cells in Culture

As early as 24 hours after establishment of the organ culture, adherentcells were visible around most sides of the tissue plug. By 36-48 hours,cells displaying a cobblestone morphology were observed. Within 7-10days of culture, evenly spaced nodules were observed, which formed acircular pattern around the plug of conjunctival tissue (FIG. 1).Initial attempts at locating goblet cells within this mixed populationof cells utilized AB/PAS (a well-documented stain for glycoconjugates infixed tissue sections). The nodules depicted in FIG. 1 reacted stronglyto AB/PAS displaying a dark blue stain indicative of acidic mucin. Tofurther purify potential goblet cells, one nodule was grown in theculture dish. All other cells were scraped from the surface of thevessel using a rubber policeman and the nodule was thoroughly rinsed torid the culture of floating, non-goblet cells. The nodule was refed withRPMI-1640 medium supplemented with 10% FBS. Within several days, gobletcells were seen leaving the nodule (FIG. 2A). Initially they assumed arounded morphology but within 7-10 days they migrated away from theparent nodule and formed circular clusters of cells, which oftenpresented a semi-cuboidal morphology (FIG. 2B). The classicalchalice-like appearance of the goblet cell was not observed in culturedcells. Often, as cells proliferated in culture, tiny droplets werevisible on the surface of cultured goblet cells suggestive of asecretory product (FIG. 2C). As these droplet-containing cells grew inculture, the droplets increased in size and number (FIG. 2D). Finally,one could see strands of fibrous material strewn over the cells (datanot shown). Upon reaching confluence, cultures were trypsinized andpassaged. The goblet cell cultures described here have been passagedthree to five times without losing morphological integrity.

These studies demonstrated that rat conjunctival goblet cells can beisolated from the fornix and the nictating membrane using a modifiedexplant culture system. Moreover, the cells can be grown and propagatedin uncoated tissue culture vessels and nourished in a basic culturemedium supplemented only with fetal bovine serum, 1-glutamine andantibiotics. Currently, RPMI medium is considered to be anon-conventional medium in the goblet cell literature. By happenstance,RPMI medium was found to be more effective than the usual types ofmediums used in other studies that failed to successfully grow isolatedgoblet cells. Cultures derived in this manner can be kept relatively(>90%) pure by scraping contaminating cell types from the culture dish.These cultured cells proliferate in vitro and can be passaged at leastthree times, with full retention of identifying cellular markers andfunctional activity.

EXAMPLE II Proliferation of Goblet Cells in Vitro

The proliferation profile of the goblet cell cultures was assessed bystaining the cells with an antibody against Ki-67 antigen, a nuclear andnucleolar protein which is exclusively expressed in proliferating cells.(Gerdes et al., 1983; Gerdes et al., 1984) All cultures were routinelyevaluated for Ki-67 reactivity. Ki-67 is localized in all primary andpassaged cultures of conjunctival goblet cells indicating that our cellsare actively proliferating in vitro. As shown in FIG. 3, greater than30% of the goblet cells in this primary culture were activelyproliferating as indicated by their reactivity to Ki-67. During thecourse of these studies, it was observed that the number of Ki-67positive cells correlated with the degree of confluence of any givenculture. As the cells approached confluence, the number of proliferatingcells declined both in primary and passaged cultures.

EXAMPLE III Characterization of Cultured Goblet Cells

Although AB/PAS was used as a screening mechanism to aid in theidentification and subsequent purification of the goblet cell cultures,it was important to determine whether or not these purified cellsretained positive reactivity to AB/PAS. The present results show thatboth primary and passaged cultures react histochemically with the stain.Shown in FIGS. 4A (rat) and 13A (human) are cells grown from arepresentative primary culture in which the cell nuclei are stained blueand displayed a red vesicle-filled cytoplasm. Further staining examinedthe reactivity of goblet cells, which appeared to contain secretorydroplets on their surface. These cells (FIG. 4B) stained a reddish colorwith no visible demarcation of the nucleus. Droplets located on top ofthe cells were bright red, indicating that these cells were associatedwith a basic type of mucin secretion product. Often, cultures appearcovered with what appeared to be a fibrous material. Upon examination,both the cells and their accompanying fibers reacted positively toAB/PAS, staining a dark blue to purple color indicative of an acidicmucin product. The reactivity of the present goblet cell cultures toAB/PAS was not lost upon subcultivation, contrary to other reports(Adams et al., 1989). AB/PAS reactivity of goblet cells in culture wascompared with that of goblet cells in vivo (FIGS. 4C (rat) and 13B(human)).

Additional histochemical verification of these goblet cell cultures wasaccomplished using a panel of lectins as histochemical probes. UEA-1recognizes the L-fucose moiety of glycoproteins in the secretorygranules of conjunctival goblet cells while HPA recognizesL-galactosamine within the secretory granules of goblet cells. BS-1,used here as a negative control, recognizes the n-galactosyl groups ofglycoproteins in stratified squamous epithelial cells. Both primary andpassaged cultures of goblet cells were labeled with anti-UEA-1 and HPA(FIG. 5) antibodies directly conjugated to a fluorophore. Goblet cellsin culture were found to react positively to UEA-1 as evidenced bycytoplasmic staining (FIG. 5A). The reactivity of cultured goblet cellsto UEA-1 was similar to that of goblet cells located in conjunctivaltissue (FIG. 5B). Similar results were observed with HPA (FIGS. 5C, 5D(rat) and 16A, 16B (human)). Both goblet cells in culture and inconjunctival tissue were found to react positively to HPA. Passagedcells also reacted positively to both UEA-1 and HPA. The reaction tothese lectins varied and appeared to be related to the levels of mucinassociated with the cell at the time it was processed for lectinhistochemistry. Goblet cells did not react with BS-1 (data not shown).

Immunocytochemical localization of the following markers was undertakento assist in the characterization of the cultured goblet cells andconsisted of the following antibodies: MUC5AC, a mucin specificallyproduced by conjunctival goblet cells (Inatomi et al.); Cytokeratin-7 anintermediate filament associated solely with goblet cells (Krenzer etal.); Muscarinic M₃ recently identified as being associated with gobletcells in the adult rat conjunctiva (Rios et al.); Cytokeratin-4,specific to the intermediate filaments found in stratified squamousepithelial cells and Muscarinic M₁ receptor subtype associated with thestratified squamous epithelial cells but not goblet cells. Primary andpassaged cultures of goblet cell cytoplasm stained intensely for MUC5ACwhile no staining was observed in the neighboring epithelial cells (FIG.6). As a positive control, goblet cells within rat conjunctival tissuestained intensely for MUC5AC (FIG. 6B).

Goblet cells in mixed cultures (FIGS. 7A (rat) and 15 (human)) and inconjunctival tissue (FIG. 7B) displayed intense fluorescence forcytokeratin-7 whereas other epithelial cell types were negative for thisintermediate filament. Conversely, when similar, mixed cultures wereanalyzed for cytokeratin-4, positive immunofluorescence was observedonly in epithelial cells migrating over the underlying goblet cellclusters (FIGS. 8A, 8B, and 14). In addition, only the M₃ receptorsubtype was positive both in cultured cells (FIG. 9A) and in gobletcells in conjunctival sections (FIG. 9B). M3 muscarinic receptors weredetected subjacent to the secretory granules of goblet cells in theconjunctiva (FIG. 9B). Several types of adjacent epithelia inconjunctival sections showed positive immunofluorescence for the M1receptor, but cultured goblet cells did not (data not shown).

When the cultured goblet cells were studied using transmission electronmicroscopy (FIGS. 10A and 10B), typical goblet cell morphology wasobserved. En-face sections of goblet cells revealed cells, whichcontained many large storage granules, with the nucleus oftenasymetrically placed.

EXAMPLE IV Mucin Secretion by Goblet Cells in Vitro

UEA-I was used to measure glycoconjugate secretion from primary andpassaged goblet cells using an ELLA (Rios et al.). Goblet cells, whichhad been in serum-free medium for 48 hours and had covered 20 and 40% ofthe surface of a 35 cm² culture dish secreted a total of 49 and 160 μgof mucin respectively. Other cultures also incubated in serum-freemedium for 72 hours which covered 10 and 256 of the same type of culturevessel were found to secrete a total of 50 and 64 micrograms of mucin,respectively. These data are shown in Table 1. TABLE 1 UEA-1-containingglycoprotein secreted by rat goblet cells % of plate Total μg of Time incovered by mucin Sample # Culture (h) goblet cells secreted 1 48 20 48.92 48 35-40 159.9 3 72 10 49.9 4 72 25 63.5

EXAMPLE V Western Blot Analysis for UEA-1 Detectable Glycoprotein andMUC5AC

Proteins from cell lysates of cultures of primary and passaged gobletcells were analyzed by western blot methods using biotinylated-UEA-1 andan antibody against MUC5AC. As shown in FIG. 11A, a high molecularweight glycoprotein of more than 220 Kda, indicative of UEA-1, waspresent in the lysate of cultured goblet cells. A similar band was alsopresent in samples of rat conjunctival homogenate, the positive controlused. Similarly, the human MUC5AC antibody reacted with a high molecularweight glycoprotein of more than 220 KDa indicating the presence ofMUC5AC (FIG. 11B) in cultured goblet cell lysate. MUC5AC was alsopresent in rat conjunctival homogenate.

EXAMPLE VI Presence of MUC5AC Glycoprotein in Human CulturedConjunctival Goblet Cells

Immunofluorescence analyses using a commercially available antibodyagainst MUC5AC glycoprotein in human conjunctival sections revealed thatMUC5AC is preferentially located in secretory granules of conjunctivalgoblet cells and not in conjunctival epithelium (FIGS. 18A, 18B, and19).

To quantify the amount of mucin secreted by cultured human conjunctivalgoblet cells an ELISA analysis was perform by using commerciallyavailable antibody MUC5AC. As revealed by ELISA analysis, MUC5ACglycoprotein was detected in the culture media in excess of 200 μg/mlwhen compared with a standard.

EXAMPLE VII Immortalizing Mammalian Goblet Cells

Mammalian goblet cells in accordance with the invention can beimmortalized using standard procedures known by those of ordinary skillin the art. By using the method of culturing goblet cells described inthe invention, an immortalized mammalian, preferably human, goblet cellline can be reproducible, while retaining the original phenotypiccharacteristics or differentiation markers of goblet cells generallyfound in vivo.

An exemplary method of producing a human immortalized goblet cell lineincludes the following protocols. Conjunctiva tissue may be obtainedfrom a human donor. The tissue is prepared for culturing using themethods described above. pL PCL telomerase (TERT) is transfected intoretroviral vectors, e.g., NIH 3T3 cells which serve as the packagingcell line, using standard procedures known in the art. Because humangoblet cells have a limited proliferative lifespan in culture,telomerase, via a retroviral vector was inserted into the culture ofhuman goblet cells. Telomerase is an enzyme that elongatesolgionucleotides from the telomere, which extends the lifespan of thegoblet cells without causing cellular tranformation and genomicinstability. These retroviral particles can introduce TERT to the gobletcells but cannot replicate inside the goblet cells due to the fact thatpL PCL hTERT does not contain the structural genes (gag, pol and env)which are needed for particle formation and replication. For instance,goblet cells are seeded into a growth medium, e.g., RPMI-1640, 12-18hours before infection with TERT. For infection, medium is collectedfrom growing packaging cells, filtered through a 0.45 micron celluloseacetate filter and applied to the goblet cells. 15 mls of packaging cellmedium is applied to human goblet cells, which have been seeded in 100mm tissue culture dishes. Following 12 hours of incubation with thepackaging cell medium, polybrene is added to the culture medium to afinal concentration of 8 micrograms per ml. Packaging cell medium isreplaced after 24 hours of incubation with medium containing puromycinor an antibiotic, where only the telomerase infected cells survive. Allnoninfected cells are removed by aspiration of the cell medium. Newmedium is added to the transfected cells, which are allowed to grow andexpand. The transfected cells are then tested for successfulimmortalization. The various studies can include, for example, testingtelomerase activity, performing their karyotype, documenting thecellular lifespan and evaluating for histochemical (AB/PAS reactivity,HPA reactivity), for immunocytochemical (cytokeratin 7, MUC5AC), forbiochemical (ability to secrete MUCSAC using ELISA protocol describedabove) and for molecular markers (PCR for MUC5AC) of human culturedgoblet cells.

In summary, conjunctival tissue was surgically removed fromSprague-Dawley rats and human patients undergoing ocular surface surgery(Massachusetts Eye and Ear Infirmary, Boston, Mass.). (Other mammalianconjunctival tissue can also be used.) For the rats, goblet cells werethen isolated from the nictating membrane and fornix using explantcultures. Human tissue were isolated from the superior fornix of theconjunctiva. Cells derived from the explants were grown and propagatedin RPMI medium supplemented with 10% fetal bovine serum andcharacterized using an enzyme-linked lectin assay (ELLA) with the lectinUlex europaeus agglutinin-1 (UEA-1), Western blot analysis, light andelectron microscopy, specialized histochemistry and indirectimmunofluorescence microscopy.

Goblet cells from mammalian conjunctiva were successfully isolated fromconjunctival explants by scraping non-goblet cells from the culturevessel. Cultures have been passaged a minimum of three to five timeswithout the loss of their specific cellular markers. The cultured cellsfulfilled the following criteria, which enabled ready identification ofthem as conjunctival goblet cells: positive staining for AB/PAS reagent,cytokeratin 7, UEA-I and HPA, MUC5AC and M₃ muscarinic receptor;negative staining for cytokeratin 4, M₁ muscarinic receptor and banderiasimplicifolia lectin. Measurements made by using the ELLA, revealedsubstantial amounts of UEA-I detectable high molecular weightglycoproteins and MUC5AC released into the medium.

Morphologically, although the cultured cells did not assume the typicalin vivo goblet cell morphology, they contained numerous secretoryvesicles, secreted droplets of mucin and as they matured in culture,strands of mucin were observed on top of the cultures. When analyzed bytransmission electron microscopy, a well-described goblet cellmorphology was observed where the cytoplasm was filled with manynumerous, translucent, distinct secretory vesicles thus supporting thefact that they are not other types of epithelia. Histochemically, thecytoplasm of the cultured cells as well as their associated mucindroplets and strands reacted with AB/PAS and with the gobletcell-associated lectins UEA-I and HPA. The immunocytochemical markerscytokeratin-7, MUC5AC and muscarinic receptor, M₃ were expressed by thecultured cells. Moreover these cells were biochemically functional invitro by retention of their ability to secrete mucin.

Proliferating goblet cells in culture, which fulfill morphological,histochemical, immunocytochemical and functional markers/functions oftheir in vivo counterparts, provide invaluable tools with which todelineate the pathobiology of the ocular surface and to study the manyfacets of goblet cell mucin synthesis and secretion in a direct,controlled and reproducible manner. Furthermore, the use of culturedgoblet cells decreases dependence on the use and/or sacrifice of largenumbers of animals to derive the same information. The availability oflarge numbers of cultured goblet cells will enable us to gain newinformation on the molecular, cellular, and functional levels,contributing to the development of novel therapies aimed at alleviatingaberrant, mucin-induced diseases of the ocular surface.

Uses

The cultured goblet cells of the invention do not only provide a goodresearch tool, they are also useful for testing the toxicity,allergenicity, tumorigenicity, intolerance, and/or other harmfuleffect(s) of various compounds, such as those used in consumer products(e.g., contact lens, shampoo, other hair care products, soaps, etc.).Additionally, the goblet cell cultures may be used for pharmaceuticaltesting, such as testing the ability of different compounds to stimulateor to inhibit goblet cell secretion, proliferation and/or otherfunction. The invention may also be used in treatment of diseasesinvolving mucin deficiency (e.g., conditions related to or caused by dryeyes). For instance, goblet cells could be transplanted to the ocularsurface of patients with decreased goblet cells and decreased mucinproduction using the methods of the present invention. The invention mayalso be used as a screening tool for, for example, to characterizedetermining laser surgery options. The present invention may be producedin a kit with instructions for experimenting the effects of mucin suchas screening various products, diagnosing mucin deficiency, studying forallergic reactivity of various foreign substances and quantitating theamount of mucin. Autoimmune effects may be deterred by using patient'sown goblet cells, from the good eye (for example), for culturing andautograft transplantation. Goblet cells, which may have been growneither on anmiotic or artificial membranes, may also be used for thedevelopment of corneal bandages for the repair of lacerated corneas or amucin deficient condition.

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While the present invention has been described in conjunction with apreferred embodiment, one of ordinary skill, after reading the foregoingspecification, will be able to effect various changes, substitutions ofequivalents, and other alterations to the compositions and methods setforth herein. It is therefore intended that the protection granted byLetters Patent hereon be limited only by the definitions contained inthe appended claims and equivalents thereof.

1. A mammalian goblet cell line that is immortalized, wherein gobletcells for said line are derived from a method of producing a culture ofgoblet cells comprising the steps of: (a) providing an explant ofconjunctival mammalian tissue; (b) culturing said explant in a growthmedium; (c) allowing said explant to grow until cell growth in the formof nodules is observed around said explant; (d) removing said explant,leaving said nodules in said growth medium; (e) allowing cells from saidnodules to grow to form said culture of goblet cells; and (f)genetically engineering cells from said culture of goblet cells to forma culture of immortalized said goblet cells.
 2. An immortalized gobletcell line.
 3. The goblet cell line of claim 1, wherein, in said methodfor deriving said cell line, said conjunctival mammalian tissue is froma human mammal.
 4. The goblet cell line of claim 1, wherein, in saidmethod for deriving said cell line, said conjunctival mammalian tissuecomprises the fornical region.
 5. The goblet cell line of claim 1,wherein, in said method for deriving said cell line, said conjunctivalmammalian tissue comprises the nictitating membrane.
 6. The goblet cellline of claim 1, wherein, in said method for deriving said cell line,said conjunctival mammalian tissue is from a mammal selected from thegroup consisting of human, rat, mouse, rabbit, cat, dog, sheep, goat,cow, and pig.
 7. A kit for examining mucin from goblet cells comprisinggoblet cells according to claim 1 or claim 2 and instructions for usethereof.